The recombinase activating genes: architects of immune diversity during lymphocyte development.

The mature lymphocyte population of a healthy individual has the remarkable ability to recognise an immense variety of antigens. Instead of encoding a unique gene for each potential antigen receptor, evolution has used gene rearrangements, also known as variable, diversity, and joining gene segment (V(D)J) recombination. This process is critical for lymphocyte development and relies on recombination-activating genes-1 (RAG1) and RAG2, here collectively referred to as RAG. RAG serves as powerful genome editing tools for lymphocytes and is strictly regulated to prevent dysregulation. However, in the case of dysregulation, RAG has been implicated in cases of cancer, autoimmunity and severe combined immunodeficiency (SCID). This review examines functional protein domains and motifs of RAG, describes advances in our understanding of the function and (dys)regulation of RAG, discuss new therapeutic options, such as gene therapy, for RAG deficiencies, and explore in vitro and in vivo methods for determining RAG activity and target specificity.

Immunological assessment of a patient with Omenn syndrome resulting from compound heterozygous mutations in the RAG1 gene.

The recombination activating gene 1 (RAG1) is essential for V(D)J recombination during T- and B-cell development. In this study, we presented a case study of a 41-day-old female infant who exhibited symptoms of generalized erythroderma, lymphadenopathy, hepatosplenomegaly, and recurrent infections including suppurative meningitis and septicemia. The patient showed a T+B-NK+ immunophenotype. We observed an impaired thymic output, as indicated by reduced levels of naive T cells and sjTRECs, coupled with a restricted TCR repertoire. Additionally, T-cell CFSE proliferation was impaired, indicating a suboptimal T-cell response. Notably, our data further revealed that T cells were in an activated state. Genetic analysis revealed a previously reported compound heterozygous mutation (c. 1186C > T, p. R396C; c. 1210C > T, p. R404W) in the RAG1 gene. Structural analysis of RAG1 suggested that the R396C mutation might lead to the loss of hydrogen bonds with neighboring amino acids. These findings contribute to our understanding of RAG1 deficiency and may have implications for the development of novel therapies for patients with this condition.

Origin and evolutionary malleability of T cell receptor α diversity.

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1-3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8-12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13-16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.

A variant of RAG1 gene identified in severe combined immunodeficiency: a case report.

BACKGROUND: The recombination-activating gene 1 (RAG1) protein is essential for the V (variable)-D (diversity)-J (joining) recombination process. Mutations in RAG1 have been reported to be associated with several types of immune disorders. Typical clinical features driven by RAG1 variants include persistent infections, severe lymphopenia, and decreased immunoglobulin levels . CASE PRESENTATION: In this study, a 2-month-24-days-old infant with recurrent fever was admitted to our hospital with multiple infections and absence of T and B lymphocytes. The infant was diagnosed with severe combined immunodeficiency (SCID). A homozygous variation c.2147G>A (NM_000448.2: exonme2: c.2147G>A (p.Arg716Gln)) was identified in the RAG1 gene using whole-exome sequencing and Sanger sequencing. The predicted 3D structure of variant RAG1 indicated altered protein stability. Additionally, decreased expression of variant RAG1 gene was detected at both the mRNA and protein levels. CONCLUSIONS: Our study identified a novel homozygous variant in RAG1 gene that causes SCID. This finding expands the variant spectrum of RAG1 in SCID and provides further evidence for the clinical diagnosis of SCID.

Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1 +/- and RAG1 -/- embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1 +/- chickens, lymphatic organs in 3-week old RAG1 -/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1+/- and RAG1-/- birds had reduced serum Igs, fewer mature CD4+ and CD8+ T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 +/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.

Different sea urchin RAG-like genes were domesticated to carry out different functions.

The closely linked recombination activating genes (RAG1 and RAG2) in vertebrates encode the core of the RAG recombinase that mediates the V(D)J recombination of the immunoglobulin and T-cell receptor genes. RAG1 and RAG2 homologues (RAG1L and RAG2L) are present in multiple invertebrate phyla, including mollusks, nemerteans, cnidarians, and sea urchins. However, the function of the invertebrates' RAGL proteins is yet unknown. The sea urchins contain multiple RAGL genes that presumably originated in a common ancestral transposon. In this study, we demonstrated that two different RAG1L genes in the sea urchin Paracentrutus lividus (PlRAG1La and PlRAG1Lb) lost their mobility and, along with PlRAG2L, were fully domesticated to carry out different functions. We found that the examined echinoid RAGL homologues have distinct expression profiles in early developmental stages and in adult tissues. Moreover, the predicted structure of the proteins suggests that while PlRAG1La could maintain its endonuclease activity and create a heterotetramer with PlRAG2L, the PlRAG1Lb adopted a different function that does not include an interaction with DNA nor a collaboration with PlRAG2L. By characterizing the different RAG homologues in the echinoid lineage, we hope to increase the knowledge about the evolution of these genes and shed light on their domestication processes.

Structural insights into the evolution of the RAG recombinase.

Adaptive immunity in jawed vertebrates relies on the assembly of antigen receptor genes by the recombination activating gene 1 (RAG1)-RAG2 (collectively RAG) recombinase in a reaction known as V(D)J recombination. Extensive biochemical and structural evidence indicates that RAG and V(D)J recombination evolved from the components of a RAG-like (RAGL) transposable element through a process known as transposon molecular domestication. This Review describes recent advances in our understanding of the functional and structural transitions that occurred during RAG evolution. We use the structures of RAG and RAGL enzymes to trace the evolutionary adaptations that yielded a RAG recombinase with exquisitely regulated cleavage activity and a multilayered array of mechanisms to suppress transposition. We describe how changes in modes of DNA binding, alterations in the dynamics of protein-DNA complexes, single amino acid mutations and a modular design likely enabled RAG family enzymes to survive and spread in the genomes of eukaryotes. These advances highlight the insight that can be gained from viewing evolution of vertebrate immunity through the lens of comparative genome analyses coupled with structural biology and biochemistry.

Guardian of the Genome: An Alternative RAG/Transib Co-Evolution Hypothesis for the Origin of V(D)J Recombination.

The appearance of adaptive immunity in jawed vertebrates is termed the immunological 'Big Bang' because of the short evolutionary time over which it developed. Underlying it is the recombination activating gene (RAG)-based V(D)J recombination system, which initiates the sequence diversification of the immunoglobulins and lymphocyte antigen receptors. It was convincingly argued that the RAG1 and RAG2 genes originated from a single transposon. The current dogma postulates that the V(D)J recombination system was established by the split of a primordial vertebrate immune receptor gene into V and J segments by a RAG1/2 transposon, in parallel with the domestication of the same transposable element in a separate genomic locus as the RAG recombinase. Here, based on a new interpretation of previously published data, we propose an alternative evolutionary hypothesis suggesting that two different elements, a RAG1/2 transposase and a Transib transposon invader with RSS-like terminal inverted repeats, co-evolved to work together, resulting in a functional recombination process. This hypothesis offers an alternative understanding of the acquisition of recombinase function by RAGs and the origin of the V(D)J system.

Inner workings of RAG recombinase and its specialization for adaptive immunity.

RAG1/2 (RAG) is an RNH-type DNA recombinase specially evolved to initiate V(D)J gene rearrangement for generating the adaptive immune response in jawed vertebrates. After decades of frustration with little mechanistic understanding of RAG, the crystal structure of mouse RAG recombinase opened the flood gates in early 2015. Structures of three different chordate RAG recombinases, including protoRAG, and the evolutionarily preceding transib transposase have been determined in complex with various DNA substrates. Biochemical studies along with the abundant structural data have shed light on how RAG has evolved from an ordinary transposase to a specialized recombinase in initiating gene rearrangement. RAG has also become one of the best characterized RNH-type recombinases, illustrating how a single active site can cleave the two antiparallel DNA strands of a double helix.

Omenn syndrome caused by a novel homozygous mutation in recombination activating gene 1.

Omenn syndrome (OS) is a type of severe combined immunodeficiency (SCID) that is distinguished by, lymphadenopathy, hepatosplenomegaly, erythroderma, alopecia with normal to elevated T-cell counts, eosinophilia, and elevated serum IgE levels. Recombination activation gene (RAG) 1 or RAG2 mutations that result in partial V(D)J recombination activity are known to be the main cause of OS. Other genes (DCLRE1C, LIG4, IL7RA, common gamma chain, ADA, RMRP, and CHD7) have also been linked to OS, although with low frequency. Here, we report a two-month-old Moroccan girl from consanguineous marriage with chronic diarrhea, recurrent and opportunistic infections, failure to thrive, desquamative erythroderma, hepatosplenomegaly, and axillary lymphadenitis. The immunological assessment showed normal lymphocyte and NK cell counts but an absence of B cells, agammaglobulinemia contrasting with a high level of IgE. On the other hand, Sanger sequencing of RAG1 and RAG2 exon 2 regions revealed a new homozygous deleterious mutation in the RAG1 gene. This c.1184C > T mutation caused a change from Proline to Leucine at position 395 of the protein, leading to a partial loss of function. Early and rapid diagnosis of the disease may facilitate urgent life-saving treatment.

Polystyrene microplastics reduce abundance of developing B cells in rainbow trout (Oncorhynchus mykiss) primary cultures.

Environmental microplastic pollution (including polystyrene, PS) may have detrimental effects on the health of aquatic organisms. Accumulation of PS microplastics has been reported to affect innate immune cells and inflammatory responses in fish. To date, knowledge on effects of microplastics on the antibody response is still very limited. Here, we investigated effects of small (0.8-20 µm) PS microplastics on the abundance of B lineage cells in primary cultures of developing immune cells from the anterior kidney of rainbow trout. Both purchased PS microbeads and PS microparticles generated from consumer products were used as microplastic sources. We first show that rainbow trout phagocytic B cells efficiently took up small (0.83-3.1 µm) PS microbeads within hours of exposure. In addition, our data revealed that PS microplastic exposure most significantly decreased the abundance of a population of non-phagocytic developing B cells, using both flow cytometry and RT-qPCR. PS microplastics-induced loss of developing B cells further correlated with reduced gene expression of RAG1 and the membrane form of immunoglobulin heavy chains mu and tau. Based on the induced loss of developing B cells observed in our in vitro studies, we speculate that in vivo, chronic PS microplastic-exposure may lead to suboptimal IgM/IgT levels in response to pathogens in teleost species. Considering the highly conserved nature of vertebrate B lymphopoiesis it is likely that PS microplastics will similarly reduce antibody responses in higher vertebrate species, including humans. Further, RAG1 provides an effective biomarker to determine effects of PS microplastics on B cell development in teleost species.

Functional regulation of an ancestral RAG transposon ProtoRAG by a trans-acting factor YY1 in lancelet.

The discovery of ancestral RAG transposons in early deuterostomia reveals the origin of vertebrate V(D)J recombination. Here, we analyze the functional regulation of a RAG transposon, ProtoRAG, in lancelet. We find that a specific interaction between the cis-acting element within the TIR sequences of ProtoRAG and a trans-acting factor, lancelet YY1-like (bbYY1), is important for the transcriptional regulation of lancelet RAG-like genes (bbRAG1L and bbRAG2L). Mechanistically, bbYY1 suppresses the transposition of ProtoRAG; meanwhile, bbYY1 promotes host DNA rejoins (HDJ) and TIR-TIR joints (TTJ) after TIR-dependent excision by facilitating the binding of bbRAG1L/2 L to TIR-containing DNA, and by interacting with the bbRAG1L/2 L complex. Our data thus suggest that bbYY1 has dual functions in fine-tuning the activity of ProtoRAG and maintaining the genome stability of the host.

Genetic identification of species and natural hybridization determination based on mitochondrial DNA and nuclear DNA of genus Zacco in Korea.

Genus Zacco specimens collected in this study were classified genetically as five species, Zacco platypus, Z. temminckii, Z. koreanus and two unidentified species, using DNA barcoding analysis based on 655 bp of mitochondrial cytochrome c oxidase subunit I (COI) gene. Two of unidentified species (Z. sp.1 and Z. sp.2) were considered to be unrecorded or new species of genus Zacco according to genetic distances between Zacco species. In addition, we determined a natural hybrid based on polymorphic base at the diagnostic positions displayed on nuclear recombination activating gene 1 (RAG1) gene (965 bp), and estimated paternal and maternal species of natural hybrid comparing phylogenetic tree between COI and RAG1, and Z. sp.1♀ × Z. koreanus♂, Z. sp.2♀ × Z. koreanus♂ and Z. koreanus♀ × Z. sp.1♂ individuals were confirmed. The habitat of natural hybrids of Z. koreanus between Z. sp.1 and Z. sp.2 was identified as Geum and Yeongsan River, respectively. In our data, only F1 hybrid generation was identified; however, generations after F1 hybrid or backcross were not demonstrated.

Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination.

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape.

Origins of the RAG Transposome and the MHC.

How innate immunity gave rise to adaptive immunity in vertebrates remains unknown. We propose an evolutionary scenario beginning with pathogen-associated molecular pattern(s) (PAMPs) being presented by molecule(s) on one cell to specific receptor(s) on other cells, much like MHC molecules and T cell receptors (TCRs). In this model, mutations in MHC-like molecule(s) that bound new PAMP(s) would not be recognized by original TCR-like molecule(s), and new MHC-like gene(s) would be lost by neutral drift. Integrating recombination activating gene (RAG) transposon(s) in a TCR-like gene would result in greater recognition diversity, with new MHC-like variants recognized and selected, along with a new RAG/TCR-like system. MHC genes would be selected to present many peptides, through multigene families, allelic polymorphism, and peptide-binding promiscuity.

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms.

The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination, allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous nonsense mutation in the 5' region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal translation initiation. Mice homozygous for the RAG1 nonsense mutation only express N-truncated RAG1 isoforms and have defects in Ag receptor rearrangement similar to human Omenn syndrome patients with truncating 5' RAG1 frameshift mutations. We show that the N-truncated RAG1 isoforms are derived from internal translation initiation start sites. Given the seemingly inactivating Rag1 mutation, it is striking that homozygous mutant mice do not have the expected SCID. We propose that evolution has garnered RAG1 and other important genes with the ability to form truncated proteins via internal translation to minimize the deleterious effects of 5' nonsense mutations. This mechanism of internal translation initiation is particularly important to consider when interpreting nonsense or frameshift mutations in whole-genome sequencing, as such mutations may not lead to loss of protein.

C-C Motif Chemokine Receptor 7 Exacerbates Hypertension Through Effects on T Lymphocyte Trafficking.

Activated T lymphocytes that infiltrate blood pressure control organs make a critical contribution to the pathogenesis of hypertension. Dendritic cells act as potent antigen-presenting cells to stimulate prohypertensive T cells. However, the mechanisms that facilitate the recruitment of prohypertensive T cells and dendritic cells into the kidney's draining lymph node during hypertension require elucidation. As CCR7 (C-C motif chemokine receptor type 7) directs the homing of lymphocytes and dendritic cells into lymph nodes, we posited that dendritic cell-mediated T lymphocyte stimulation in the renal lymph node is CCR7 dependent and required for a full hypertensive response. We found that CCR7-deficient (CCR7 KO) mice had a blunted hypertensive response in our model of chronic Ang II (angiotensin II) infusion. Ang II-infused CCR7 KO animals had exaggerated accumulation of CD8+ T cells in the kidney but reduced numbers of CD4+ and CD8+ T cells in the kidney's draining lymph node. To understand whether CCR7-dependent homing of T lymphocytes or dendritic cells into the lymph node regulates the hypertensive response, we injected CCR7 KO or wild-type T cells or dendritic cells into CCR7 KO recipients, neither of which restored the full hypertensive response to Ang II infusion. However, adoptive transfer of wild-type but not CCR7 KO T lymphocytes into RAG1 (recombination-activating gene 1)-deficient mice that lack a lymphocyte niche restored full blood pressure elevation during Ang II infusion. Thus, CCR7-dependent interactions between T lymphocytes and dendritic cells are essential for T lymphocyte stimulation and hypertension accruing from inappropriate activation of the renin-angiotensin system.

Comparative genetic stock structure in three species of commercially exploited Indo-Malay Carangidae (Teleosteii, Perciformes).

We examine genetic structuring in three commercially important species of the teleost family Carangidae from Malaysian waters: yellowtail scad Atule mate, bigeye scad Selar crumenophthalmus and yellowstripe scad Selaroides leptolepis, from the Indo-Malay Archipelago. In view of their distribution across contrasting habitats, we tested the hypothesis that pelagic species display less genetic divergence compared with demersal species, due to their potential to undertake long-distance migrations in oceanic waters. To evaluate population genetic structure, we sequenced two mitochondrial (mt)DNA [650 bp of cytochrome oxidase I (coI), 450 bp of control region (CR)] and one nuclear gene (910 bp of rag1) in each species. One hundred and eighty samples from four geographical regions within the Indo-Malay Archipelago including a population of yellowtail from Kuwait were examined. Findings revealed that the extent of genetic structuring among populations in the semi-pelagic and pelagic, yellowtail and bigeye were lower than demersal yellowstripe, consistent with the hypothesis that pelagic species display less genetic divergence compared with demersal species. The yellowtail phylogeny identified three distinct clades with bootstrap values of 86%-99% in mtDNA and 63%-67% in rag1. However, in bigeye, three clades were also observed from mtDNA data while only one clade was identified in rag1 dataset. In yellowstripe, the mtDNA tree was split into three closely related clades and two clades in rag1 tree with bootstraps value of 73%-99% and 56% respectively. However, no geographic structure appears in both mtDNA and rag1 datasets. Hierarchical molecular variance analysis (AMOVA), pair wise FST comparisons and the nearest-neighbour statistic (Snn ) showed significant genetic differences among Kuwait and Indo-Malay yellowtail. Within the Indo-Malay Archipelago itself, two distinct mitochondrial lineages were detected in yellowtail suggesting potential cryptic species. Findings suggests varying degrees of genetic structuring, key information relevant to management of exploited stocks, though more rapidly evolving genetic markers should be used in future to better delimit the nature and dynamics of putative stock boundaries.

Sympatric lineages in the Mantidactylus ambreensis complex of Malagasy frogs originated allopatrically rather than by in-situ speciation.

Madagascar's biota is characterized by a high degree of microendemism at different taxonomic levels, but how colonization and in-situ speciation contribute to the assembly of local species communities has rarely been studied on this island. Here we analyze the phylogenetic relationships of riparian frogs of the Mantidactylus ambreensis species complex, which is distributed in the north of Madagascar and was originally described from Montagne d'Ambre, an isolated mountain of volcanic origin, currently protected within Montagne d'Ambre National Park (MANP). Data from mitochondrial DNA, and phylogenomic data from FrogCap, a sequence capture method, independently confirm that this species complex is monophyletic within the subgenus Ochthomantis, and identify two main clades within it. These two clades are separated by 5.6-6.8% pairwise distance in the mitochondrial 16S rRNA gene and co-occur in MANP, with one distributed at high elevations (940-1375 m a.s.l.) and the other at lower elevations (535-1010 m a.s.l.), but show almost no haplotype sharing in the nuclear RAG1 gene. This occurrence in syntopy without admixture confirms them as independent evolutionary lineages that merit recognition as separate species, and we here refer to them as high-elevation (HE) and low-elevation (LE) lineage; they will warrant taxonomic assessment to confidently assign the name ambreensis to one or the other. Populations of the M. ambreensis complex from elsewhere in northern Madagascar all belong to the LE lineage, although they do occur over a larger elevational range than in Montagne d'Ambre (285-1040 m a.s.l.). Within LE there are several phylogroups (LE1-LE4) of moderately deep divergence (1.5-2.8% in 16S), but phylogroup LE4 that occurs in MANP has a deeply nested phylogenetic position, as recovered separately by mitochondrial and sequence capture datasets. This suggests that HE and LE did not diverge by a local fission of lower and upper populations, but instead arose through a more complex biogeographic scenario. The branching pattern of phylogroups LE1-LE4 shows a clear south-to-north phylogeographic pattern. We derive from these results a testable hypothesis of vicariant speciation that restricted the HE lineage to MANP and the LE candidate species to a climatic refugium further south, with subsequent northwards range expansion and secondary colonization of MANP by LE. These results provide an example for complex assembly of local microendemic amphibian faunas on Madagascar.